The neural cell adhesion molecule, NCAM, mediates cell-cell adhesion via homophilic (NCAM-NCAM) binding. NCAM plays a key role in neural development, neuronal differentiation and synaptic plasticity, including learning and memory consolidation.
Intercellular interactions play a crucial role in a wide range of biological processes, including cell migration, survival and differentiation. These phenomena depend upon protein recognition at the cell surface mediated by cell-cell adhesion molecules (CAMs).
The neural cell adhesion molecule, NCAM, originally described as a synaptic membrane protein (Jorgensen and Bock, 1974), and later shown to mediate cell-cell adhesion was the first mammalian cell adhesion molecule identified. NCAM belongs to the immunoglobulin (Ig) superfamily. Alternative splicing of mRNA and post-translational modifications generate a large number of NCAM isoforms. The three major NCAM isoforms have identical extracellular parts consisting of five Ig modules and two fibronectin type III modules.
NCAM is known to mediate Ca2+-independent cell-cell and cell-substratum adhesion via homophilic (NCAM binding to NCAM) and heterophilic (NCAM binding to other molecules) interactions (Berezin et al., 2000). The different modules of NCAM have been shown to perform distinct functions. NCAM binds various extracellular matrix components such as heparin/heparan sulfate, chondroitin sulfate proteoglyeans, and different types of collagen. The heparin binding sequence is localized to the Ig2 module. NCAM also binds to the neural cell adhesion molecule L1. This interaction is believed to take place between the fourth Ig module of NCAM and an oligomannosidic moiety expressed on L1.
Despite extensive studies, the precise mechanism of the homophilic binding of NCAM remains unclear, and the published results are to some extent contradictory. NCAM homophilic binding was originally reported to depend on an antiparallel interaction between Ig3 modules from two opposing NCAM molecules. Cell aggregation experiments performed on mouse L-cells expressing chicken NCAM with deletions of different Ig modules indicated an involvement of the Ig3 module. Later, employing microspheres coated with individual recombinant Ig modules of chicken NCAM, binding was demonstrated between the Ig1 and Ig5 modules, and between the Ig2 and Ig4 modules, whereas microspheres coated with Ig3 exhibited strong self-aggregation (Ranheim et al., 1996). However, a study by Atkins et al. (2001) on the solution structure of the Ig3 module of chicken NCAM including ultracentrifugation experiments did not support the suggested dimerization of Ig3.
A binding between recombinant modules of rat Ig1 and Ig2 was demonstrated by means of surface plasmon resonance analysis (Kiselyov et al., 1997). The three-dimensional structures of individual modules of rat Ig1 and Ig2, and the chicken Ig1 module, have been determined by nuclear magnetic resonance (NMR) spectroscopy, resulting in the identification of amino acid residues involved in the homophilic binding between the Ig1 and Ig2 modules (Thomsen et al., 1996; Jensen et al., 1999; Atkins et al., 1999). The crystal structure of the Ig1-2 fragment of rat NCAM provided detailed information on the cross-like Ig1-2 dimer, and pointed out the key residues in this interaction, namely F19 and Y65 (Kasper et al., 2000). Recently, it was demonstrated that a point mutation of F19 (F19S) did not affect cell aggregation mediated by full length NCAM, even though it abolished dimerization of the Ig1-2-3 fragment, which otherwise takes place in solution (Atkins et al., 2001). These results therefore question the suggested Ig3-to-Ig3 (Rao et al., 1992; Ranheim et al., 1996) and Ig1-to-Ig2 (Kiselyov et al., 1997; Kasper et al., 2000) models of NCAM homophilic binding.
Thus, two non-overlapping homophylic binding sites of NCAM have been described in scientific literature: the Ig3-to-Ig3 and Ig1-to-Ig2 binding sites. The sequences derived from these two sites have been shown to be capable of stimulating neurite outgrowth and modulating adhesion of neural cells (WO03020749, Soroka et al, 2002; Rao et al., 1992; Ranheim et al., 1996). It has also been shown that peptide sequences, which are capable to bind to the Ig3-to-Ig3 binding site, do not interfere with the biological effects mediated by the Ig1-to-Ig2 binding site, and vice versa. The latter finding indicate that NCAM homophylic adhesion has a much more complex mechanism, then just the mechanism of mechanistic binding of two individual NCAM molecules though the multiple homophylic binding sites, and that the involvement of one or another homophylic binding site in a process mediated by NCAM may depend on a particular NCAM environment, such as for example the presence of a ligand of one or another binding site, or availability of one or another site for binding.
The present invention provides a method of modulating such processes by providing compounds capable of binding to NCAM modules Ig1, Ig2 and/or Ig3 through a novel homophylic binding site.